Preparation of microparticles for controlled release of water-soluble substances

ABSTRACT

The subject of the present invention is a process for preparing microparticles of the microsphere type of a water-soluble substance and a biocompatible and biodegradable polymer controlling the kinetics of release of the said substance consisting of a matrix of the said polymer within which the said water-soluble substance is regularly dispersed, characterized in that 
     a) the said polymer is dissolved in a first volatile organic solvent immiscible with water, 
     b) the said water-soluble substance is dissolved in a second solvent which is miscible with the said first solvent, is a solvent for the polymer, and miscible with water, 
     c) the solution of the said substance and the solution of the said polymer are mixed, 
     d) an organic phase of the polymer and of the said substance is obtained which is then emulsified in an immiscible dispersant medium consisting of an aqueous phase containing an emulsifying agent, 
     e) the two solvents are then removed from the microspheres being formed with stirring, the first solvent being removed by evaporation, the second solvent and part of the first solvent which is miscible therewith being removed by passage towards the aqueous phase by a mechanism of phase separation, 
     f) after removal of the solvents, the microspheres formed are recovered, optionally after washing in water and sieving.

This application is a continuation of United States application Ser. No.07/810,403 filed as PCT/EP91/00307, Feb. 18, 1991, now abandoned.

The present invention relates to microparticles of the microsphere typeconsisting of a matrix of biocompatible and biodegradable polymer withinwhich a water-soluble substance, in particular a polypeptide isdispersed, the polymer controlling the kinetics of release of the saidsubstance, such that the said substance is progressively released over adefined period of time when the microparticles are placed in an aqueousmedium, in particular of the physiological type.

The present invention furthermore relates to a process for preparing thesaid microparticles.

Such microspheres were initially developed for contraception using.steroid hormones (see in particular U.S. Pat. No. 3,773,919).

A difficulty with inventions in this area is the need to determine thepolymers exhibiting defined properties as a function of the type ofactive ingredient to be incorporated, taking into account, on the onehand, its structure and its chemical properties and, on the other hand,the desired kinetics of release. It is also necessary to determine, fora given type of substance, the rate of release and the level of chargeensuring an optimum therapeutic protection for a chosen period when thesaid substance is a medicinal product.

Furthermore, certain limitations may occur in the preparation process inthe technique for incorporating the medicinal product into the polymerwhich depends on the polymer/medicinal product pair, in particular onthe optional existence of a common solvent for the components, theexistence of thermal properties of the latter, their miscibility and thelike. Moreover, this incorporation must not affect the properties of theactive ingredient.

Originally, the aim of the present invention was the preparation ofmicrospheres which would contain a water-soluble polypeptide and inparticular calcitonin, and would release the latter in a progressive andcontinuous manner for a period of at least 8 days and even 30 days forcalcitonin once it is injected in man.

The incorporation of active ingredients inside polymer biomaterials inthe form of microspheres is performed by the so-calledmicroencapsulation techniques. Microencapsulation includes all thetechniques enabling the preparation of individualised particles of themicrosphere type whose size ranges from 1 to 250 μm.

Microencapsulation processes are traditionally classified into twogroups.

1. So-called microencapsulation by coacervation or by phase separation

First, the product to be encapsulated is dispersed in the solution of apolymer intended to subsequently form the matrix of the microcapsules.Secondly, the coacervation of the polymer is induced by aphysico-chemical modification of the reaction medium, in particular bymeans of a phase separation inducing agent. Thirdly, coacervate dropletsthat form around the material to be encapsulated are stabilized andsolidified by means of a nonsolvent of the polymer, for example heptane.

The main disadvantage of this method is the use of large amounts ofsolvents with, in addition to cost constraints, problems of toxicitylinked to the solvents, such as heptane, used. This is because thetechniques by coacervation using heptane do not enable its completeremoval. A large amount of residual solvents, of the order of 5 to 10%of heptane, is observed in the microspheres.

Independently of the above, it has also been observed that aggregates ofmicrospheres causing a high loss of yield in the production of thesemicrospheres by this method and sometimes requiring the total rejectionof some batches which have thus become unusable, were often produced.The tendency of the microspheres to aggregate causes additionaldifficulties at the time of suspending the microspheres for injection,in the case of injectable microspheres.

Another disadvantage of the technique by phase separation is thenonhomogeneous distribution of the active substance in the microsphereswith irregular release, and in general a first phase of acceleratedrelease ("burst effect"). This is observed in particular when the activesubstance is suspended in the polymer solution, in particular because itis not soluble in the solvent for the polymer. This generally applies,for example, to polypeptides.

2. Microencapsulation by solvent evaporation

The so-called process by solvent evaporation-(or alternatively byemulsion/evaporation) also leads to the production of microspheres. Theactive ingredient to be encapsulated is traditionally dispersed in asolution of polymer in a volatile organic solvent. This phase isemulsified by means of a surface-active agent in a non-miscibledispersing medium (water or mineral oil). The organic solvent evaporateswith stirring. After the evaporation, the microspheres are recovered byfiltration or centrifugation.

The advantages of the technique by emulsion/evaporation are the absenceof toxic solvents such as heptane, and the absence of agglomeration ofthe microspheres.

This technique by emulsion/evaporation is a technique which is simpler,more flexible and easier to industrialize than the technique by phaseseparation or coacervation. It makes it possible to use reduced amountsof solvent.

It offers a great freedom of action on the various parameters such asthe size of the microspheres, the rate of release and the level ofcharge of the active substance.

Traditionally, this technique is primarily applied to the encapsulationof lipophilic substances such as steroids and nitrosoureas. Themicroencapsulation of hydrophilic active ingredients requires the use ofan apolar dispersing phase such as a mineral oil. Acetone/paraffinsystems are conventionally used. However, the levels of incorporation ofthe hydrophilic active ingredient into the microspheres relative to theamounts employed in the process are fairly low and, moreover, thissystem involves a limitation with respect to the types of polymers whichmay be used given that it requires the polymer to be soluble in acetone,which is the case with lactic acid polymers, but which is not the casefor lactic acid and glycolic acid copolymers.

This technique by emulsion/evaporation is therefore traditionallyrecognised as unsuitable for water-soluble peptides and for allwater-soluble substances.

The method by phase separation or coacervation is therefore the methodselected as being the most adapted to water-soluble substances, inparticular to water-soluble polypeptides.

In order to try to obviate the disadvantages of the method byemulsion/evaporation, a method by double emulsion (water/oil/water) hasbeen described in European Patent Application EP 190,833. It is,however, complicated, relatively difficult to industrialize and does notmake it possible to obtain the desired kinetics of release according tothe present invention.

The object of the present invention is to provide microparticles of themicrosphere type for the progressive release of a water-soluble activesubstance such as a medicinal product, in particular a water-solublepolypeptide, dispersed in a biocompatible and biodegradable polymermatrix enabling a release of the active substance in particular over aperiod of at least 8 days, and this in a regular manner with inparticular the absence of a first phase of accelerated release ("bursteffect").

More particularly, another object of the present invention is to enablethe progressive release of calcitonin over a period of about 30 days inthe same conditions as mentioned above.

Another object is to provide a process for preparing these microspheres,economically and industrially acceptable with in particular asufficiently high level of incorporation of the active substance to beincorporated in microsphere form.

Another object is to provide microspheres and a process of preparationsuch that the residual level of solvents, in particular toxic solventsis very low.

According to the present invention, a novel process has been discoveredwhich makes it possible to adapt the technique by emulsion/evaporationto water-soluble substances and in particular to water-solublepolypeptides with a level of incorporation of the substance into themicrospheres greater than 90% and a very low level of residual solvent.

In the present application, "level of incorporation" is understood asmeaning the level of substance which is found in the microspheres at theend of the process relative to the amount of substance initiallyemployed in the process.

Contrary to the methods described in the literature in which the activesubstance to be incorporated is a peptide which is placed inmicroparticulate suspension in the polymer solution, according to theinvention, the active peptide substance is first solubilized in asuitable solvent whose characteristics are indicated below.

The subject of the present invention is therefore a process forpreparing microspheres of a water-soluble substance, in particular of awater-soluble polypeptide and a biocompatible and biodegradable polymercontrolling the kinetics of release of the said substance according towhich:

a) the polymer is dissolved in a first volatile organic solventimmiscible with water,

b) the water-soluble substance is separately dissolved in a second,non-volatile solvent which is miscible with the first solvent, which isalso a solvent for the polymer, and which is miscible with water (thesaid second solvent therefore in fact constitutes a third solventbetween water and the first solvent which is both a solvent for thepolymer and a solvent for the water-soluble substance),

c) the polymer solution obtained in stage a) and the solution of theactive substance obtained in stage b) are mixed,

d) an organic phase of the polymer and of the active substance isobtained which is then emulsified in an immiscible dispersant mediumconsisting of an aqueous phase containing an emulsifying agent,

e) the two solvents are then removed from the microspheres being formed,with stirring, the first solvent being removed by evaporation, thesecond solvent as well as part of the first solvent which is miscibletherewith passing into the aqueous phase by a mechanism of phaseseparation,

f) after removal of the solvents, the microspheres formed are recovered,optionally after washing in water and sieving.

Furthermore the microspheres are then preferably treated so as to ensuretheir preservation.

Volatile organic solvent is understood as meaning an easily evaporablesolvent, that is to say evaporable with stirring at room temperature andreduced pressure under air suction.

The second solvent is put in a proportion relative to the first solventsuch that there is no precipitation of the polymer in the aqueous phase.This is because if the amount of the said second solvent is too high,the emulsion is no longer obtained, the polymer phase and the aqueousphase become miscible and there is precipitation of the polymer in theaqueous phase. The emulsion remains possible with ratios of secondsolvent to first solvent ranging up to 70/30 by volume. The minimumproportion of the second solvent relative to the first solvent is ofcourse linked to the level of charge which it is desired to obtain inthe case of the active substance in the microsphere. The proportion of5/95 can be given as a minimum value.

There may be mentioned with no limitation being implied, by way of firstsolvent, chloroform or dichloromethane and, by way of second solvent,dimethylacetamide DMA, tetrahydrofuran THF, dioxane, DMSO(dimethylsulfoxide) or DMF (dimethylformamide).

In a preferred embodiment of the process according to the invention, thepolymer is dissolved in dichloromethane and the water-soluble substanceis dissolved in dimethylacetamide (DMA).

The use of the third solvent, which is both a solvent for thewater-soluble substance and is miscible with the solvent for thepolymer, ensures a homogeneous distribution of the active substance inthe polymer phase. However, the advantage of using the third solvent isnot, as seen, limited to the mere solubilization of the activesubstance. Given its miscibility with the aqueous phase, it alsocontributes to the incorporation process of the active substance intothe microsphere.

Thus, contrary to the conventional method by emulsion/evaporation wherethe hardening of the microsphere is carried out solely by simple andslow evaporation of the polymer solvent such as CH₂ Cl₂, in the methodaccording to the present invention, the phenomenon is linkedsimultaneously to the evaporation of the said first solvent such as CH₂Cl₂ and to a rapid phase separation of the second solvent such as DMAtowards the aqueous phase. Through the miscibility of the two solvents,some of the first solvent also accompanies the second towards theaqueous phase which considerably reinforces and accelerates itsextraction. The method thus combines the processes of evaporation andphase separation by substantially modifying the kinetics of extractionof the solvents as compared with a traditional method ofemulsion/evaporation. The kinetics of the incorporation process istherefore modified, which contributes, with the homogeneity of thedistribution, to the high level of incorporation observed, as will bedescribed later.

One of the advantages of the process according the invention is toprovide microspheres whose residual content of toxic solvent, such asdichloromethane, is very low (in particular lower than 1.5%).

In an improved embodiment of the process according to the inventionparticularly useful when the said first solvent for the polymer is atoxic solvent, the level of residual solvent may be reduced if theaqueous dispersant phase comprises a third nontoxic solvent misciblewith water and miscible with the said first solvent for the polymer.This third solvent is added to the aqueous dispersant phase of theemulsion before the onset of the formation of this emulsion. Thisaddition of such a third solvent makes it possible to promote theremoval of the first solvent for the polymer by a better extraction ofthe latter towards the aqueous phase with which it is otherwiseimmiscible. The residual level the solvent for the polymer in themicrospheres is thus thereby reduced.

The said third solvent is added to the aqueous phase in proportionswhich continue to make possible the emulsion and the dissolution of theemulsifying agent in this same aqueous phase.

When the solvent for the polymer is dichloromethane, ethanol isadvantageously used as said third solvent in a proportion ranging up to20% by volume of the aqueous phase. The residual level ofdichloromethane in the microspheres is thereby considerably reduced, forexample to 0.2% relative to the dry weight of the microspheres.

In a specific embodiment of the invention, the water-soluble substanceis a polypeptide or one of its pharmaceutically acceptable salts and thepolymer is a polymer resulting from the condensation of monomers chosenfrom alpha-hydroxycarboxylic acids and lactones. Preferably, the polymeris a lactic acid (alpha-hydroxypropionic acid) and glycolic acid(alpha-hydroxyacetic acid) copolymer hereafter abbreviated PLGA, invariable proportions between the lactic acid and glycolic acid units, ora mixture of such polymers.

These polymers are well known in the state of the art. They are known togenerate products naturally present and metabolized in the organism andare therefore considered as biodegradable and free from substantialtoxicity in the normal conditions of use. They are, moreover, soluble indichloromethane.

DMA exhibits all the required characteristics as second solvent giventhat the polymers mentioned above and, in particular the lactic acid andglycolic acid copolymers are soluble therein. DMA is not easilyevaporable contrary to dichloromethane. On the other hand, it may beextracted by the aqueous phase with which it is miscible, optionallyduring the evaporation of the dichloromethane.

In a specific embodiment of the invention, a lactic acid and glycolicacid copolymer (PLGA) which preferably comprises a proportion betweenthe lactic acid and glycolic acid units ranging from 40:60 to 75:25respectively, is used.

The molecular mass of the polymer will preferably be between 20,000 and100,000. The water-soluble polypeptides according to the invention havea molecular mass normally between 200 and 100,000.

Thus, the process according to the invention is applicable to the mostdiverse polypeptides such as hormones, cytokines, growth factors,neuropeptides, coagulation factors, enzymes, anti-enzymes, solublereceptors or biologically active derivatives of these substances. Theremay also be mentioned, in particular, with no limitation being implied,human growth hormone (HGH), bovine growth hormone (BGH), porcine growthhormone (PGH), ovine growth hormone and the like) somatomedins, IGF-1,IGF-2 and insulins; LH-RH and its analogues, GH-RP and its analogues;somatostatin and its analogues; ACTH, ADH, PTH, PTH 1-34, CRH, GH-RH,inhibit, activin, motilin, relaxin, ANF, endothelins, their derivativesand analogues; IL1, IL2, IL3, IL4, IL5, IL6, IL7, IL8, IL9, G-CSF,GM-CSF, M-CSF, alpha, beta and gamma interferons, alpha and beta TNF,alpha and beta TGF, PDGF, EGF, FGF, osteogenins, their analogues andderivatives; TPA, factor VIII, yon Willebrandt factor, hirudin,echistatin, bitisatin, alpha-1-antitrypsin, superoxide dismutase,soluble CD4 and their analogues and derivatives; peptide derivatives oranalogues of adhesion molecules, of lymphocyte receptors or ofcomponents of the major histocompatibility complex; and calcitoninswhether they are obtained from man, pig, salmon, eel or from anotherspecies, as well as their analogues and derivatives.

According to the process of the invention, the level of charge ofsubstance in the polymer may rise from 0.01 to 40%. Level of charge isunderstood as meaning the ratio by weight of the substance to the weightof the polymer in the microsphere. Generally, this level of charge willpreferably be from 1 to 10% depending on the desired therapeuticprotection.

Thus, in the case of calcitonin, its solubility in DMA which is 50mg/mlmakes it possible to envisage a level of charge ranging up to 31.5% ofsalmon calcitonin in 50:50 PLGA microspheres taking into account themaximum DMA/CH₂ Cl₂ proportion of 70/30 which may be used. However, thebest therapeutic protection is obtained with a level of charge of only 1to 2% in the case of salmon calcitonin, and 10% in the case of humancalcitonin which is less active. It is therefore clear that the processaccording to the invention produces no practical limitation with respectto the level of charge of the substance in the microsphere.

Water-soluble polypeptide is understood, in the present application, asmeaning a solubility of not less than 0.1 mg/ml in water.

When the solubility of the polypeptide is too low for the level ofcharge which it is desired to obtain in the microsphere taking intoaccount the target therapeutic protection, a mixture of DMA and watermay be used as second solvent, it being possible for the amount of waterin proportion by volume relative to the DMA to rise up to 50%.

According to the invention, a ballasting substance may be added into thepolymer phase at the same time as the active substance in order tofacilitate the release by accelerating and regulating the kinetics ofrelease. This ballast may be either dissolved or suspended in thepolymer phase. The most diverse ballasts may be used such as proteinslike HSA, gelatin, sugars, polysaccharides, amino acids or polypeptides.The level of charge of ballast may reach 5%.

In the process according to the invention, preferably, theemulsification is carried out with vigorous stirring so as to obtainmicroparticles less than 200μ in size, preferably between 25 and 200μ.Thus, injectable microspheres are obtained when they are formulated intoan injectable solute.

This is because the size of the microspheres depends on the force of thestirring and in particular the speed imposed on the helix during theemulsion (if it increases, the size decreases). It also depends on theconcentration of emulsifier in the aqueous phase (if it increases, thesize decreases), and finally, of course, on the size of the sieve mesh.A maximum distribution of the mean size of the microspheres between 25μand 200μ is easily and reproducibly obtained by acting on theaforementioned parameters.

Gelatin, carboxymethylcellulose, methylcellulose, hydroxypropylcellulose, hydroxyethyl cellulose, polyvinyl alcohol (PVA) oralternatively nonionic detergents such as Tween 80 or ionic detergentsmay be mentioned by way of emulsifying agent present in the aqueousphase, with no limitation being implied.

The concentration of the emulsifier in the aqueous phase influences thesize of the microspheres. The latter increases as the concentration ofemulsifier decreases. According to the invention, the concentration ofemulsifier in the aqueous phase may range from 0.1 to 10%(weight/volume).

In a specific embodiment, gelatin, which is particularly well acceptedas a product which can be injected into the organism has been used asemulsifying agent.

The process according to the invention offers the usual advantages of anemulsion/evaporation technique as compared with the phase separation orcoacervation technique:

absence of toxic solvent other than the solvent for the polymer,

absence of agglomeration of microspheres (easier industrialization andinjection),

absence of handling of a large amount of solvent during the industrialproduction,

possibility of acting on various parameters in order to easily modulatethe kinetics of release as a function of the therapeutic applicationconcerned: the characteristics of the polymer, the presence of ballast,the size of the microspheres, the level of charge of water-solublesubstance.

The other advantages specific to the use of a third solvent forsolubilizing the water-soluble substance are:

a level of incorporation of the substance which may be higher than 90%,

the regular and homogeneous distribution of the water-soluble substancesolubilized in the polymer, and in the final microsphere,

the possibility of achieving substantial levels of charge ofwater-soluble substance while preserving a high level of incorporation,

a reduction of the residual level of the solvent for the polymer in thefinal product,

the absence or the reduction of "burst" effect during the kinetics ofrelease of the substance.

The subject of the present invention therefore is also microparticles ofthe microsphere type for the progressive release of a water-solubleactive substance such as a medicinal product, in particular of awater-soluble polypeptide regularly distributed within a matrix of abiocompatible and biodegradable polymer which is soluble in an organicsolvent which is immiscible water, enabling a release of the said activesubstance over a period of at least 8 days, and this in a regular mannerwith in particular the absence of a first phase of accelerated release.

According to the invention, the microspheres exhibit a residual level ofsolvent lower than 1.5%, preferably lower than 0.5% (w/w) and inparticular are free from heptane. In effect, the level of DMA is lowerthan 0.5%, and in general even of the order of 0.1% (w/w) and the levelof CH₂ Cl₂ is generally lower than 0.5%, and even often lower than 0.2%(w/w). Consequently, one of the characteristics of the microspheresaccording to the present invention is to have a level of residual toxicsolvent lower than 0.5%, preferably lower than 0.2% by weight relativeto the dry weight of the microspheres.

The polymer will advantageously be a dichloro-methane-soluble polymer,in particular a lactic acid and glycolic acid copolymer.

The microspheres obtained according to the invention are moreovercharacterized by the homogeneous distribution of the active substancewhich manifests itself by the high reduction of the burst effect, by alow tendency to agglomerate which distinguishes them from themicrospheres obtained by coacervation which is usually described for theencapsulation of peptides, and by substantially reduced residual levelsof solvents.

Other characteristics and advantages of the present invention willemerge from the following examples:

EXAMPLE 1 Preparation of Calcitonin Microsphere in a Matrix of LacticAcid and Glycolic Acid Copolymer

5 g of 50/50 PLGA (lactic acid and glycolic acid copolymer: 50/50) aredissolved in 40 g of dichloromethane CH₂ Cl₂.

5 mg of salmon calcitonin are dissolved in 5 ml of dimethylacetamide(DMA). The solubilized peptide is mixed with the polymer phase. 150 mlof 10 mM phosphate buffer at pH 8 containing 4% (weight/volume) ofgelatin are added to this mixture.

An emulsion is produced by mixing the polymer/calcitonin phase with theaqueous phase containing the gelatin by means of a helix revolving at1,500 revolutions/minute for 1 minute.

The evaporation of the solvent for the polymer CH₂ Cl₂ is carried out bymaintaining a stirring of 500 revolutions/minute in the emulsifiedmixture. The extraction of the DMA towards the aqueous phase isperformed in parallel. This evaporation is carried out at roomtemperature with air suction until complete evaporation of the solvent.

The emulsified mixture is then diluted two-fold with water and stirred 1hour at 37° C.

The microspheres thus obtained are then sieved in water in order toselect those having a suitable size in order to be injected, that is tosay between 25 and 200μ.

The sieved microspheres are then dried under vacuum at room temperature.

After drying, the microspheres are fully individualized and are easilyresuspended in a physiological medium for injection. Microscopicexamination confirms the homogeneity and the complete absence ofagglomeration of the microspheres.

The levels of residual solvents in the microspheres are:

solvent for the polymer CH2Cl₂ : lower than 0.5% relative to the dryweight of the microspheres,

third solvent DMA: lower than 0.1% relative to the dry-weight of themicrospheres

EXAMPLE 2 Modification of the Extraction of the Solvent for the Polymerby Using the Third Solvent

This example illustrates an improvement of the removal of the firstsolvent (CH₂ Cl₂) by the mechanism of phase separation by virtue of thepresence of the second solvent (DMA).

Materials

Polymer phase:

50/50 PLGA η=0.8: 2.5 g

salmon CT : 5 mg level of charge of 0.2%/PLGA.

    ______________________________________                                        A                  B                                                          ______________________________________                                        CH.sub.2 Cl.sub.2 volume/DMA                                                                     CH.sub.2 Cl.sub.2 volume/DMA                               volume = 9/1       volume = 7/3                                               CH.sub.2 Cl.sub.2 = 20.25 g                                                                      CH.sub.2 Cl.sub.2 = 15.75 g                                DMA = 2.25 ml      DMA = 6.75 ml                                              ______________________________________                                    

Aqueous phase:

10 mM phosphate buffer, pH 8 with 4% gelatin as emulsifier: 75 ml.

Method

The peptide is solubilized in the volume of DMA used in the reaction.

This solution is then added to the PLGA solubilized in CH₂ Cl₂.

The aqueous phase is mixed with this PLGA phase by means of a helixrevolving at 1,500 r/min for 1 minute.

A stirring of 500 r/min is maintained for 12 hours at room temperaturewith air suction at the surface of the reaction medium.

The microspheres are then sieved and washed in water.

They are then dried in an oven at room temperature under vacuum.

Results

The residual level of CH₂ Cl₂ in these microspheres is measured by gaschromatography.

    ______________________________________                                                             A      B                                                 ______________________________________                                        CH.sub.2 Cl.sub.2 volume/DMA volume                                                                  9/1      7/3                                           Residual level of CH.sub.2 Cl.sub.2                                                                  2.58%    0.44%                                         (%)                                                                           (CH.sub.2 Cl.sub.2 weight/microsphere weight)                                 ______________________________________                                    

EXAMPLE 3 Level of Incorporation of Calcitonin and "Burst" Effect

The microspheres obtained by the process according to the invention arecharacterized by a reduction of the "burst effect" and a highly improvedlevel of incorporation compared to the conventional process byemulsion/evaporation.

The table below presents the results for the preparations obtained withthe conventional method by emulsion/evaporation compared to the methodaccording to the invention for various polymers.

The presence of a "burst" effect essentially reflects nonhomogeneity ofthe distribution of the active ingredient in the PLGA matrix.

In the "conventional" method by emulsion/evaporation followed and themethod according to the invention, 2.5 g of polymer dissolved in 20 g ofCH₂ Cl₂ are used. 5 mg of salmon calcitonin are used and 10 mg ofgelatin as ballast.

In the "conventional" method, the calcitonin and the ballast aresuspended in the polymer phase. In the method according to theinvention, the calcitonin is dissolved in 2.5 ml of DMA to which theballast is added in the form of a 5% aqueous solution of gelatin (0.2ml). This calcitonin-ballast-DMA mixture is then added to the polymerphase so as to form a single homogeneous phase.

In the methods, the aqueous phase consists of 75 ml of 4% gelatin inwater (weight/volume). The two phases are emulsified by stirring with ahelix at 1,500 revolutions/minute for i minute. A stirring of 500revolutions/minute is maintained, on the one hand, in order tofacilitate the evaporation of the CH₂ Cl₂ and, on the other hand, in themethod according to the invention, to enable the DMA to be extracted inthe aqueous phase, in both cases, the microspheres are washed in waterand collected on sieves.

    ______________________________________                                                               Emulsion/                                                        "Conventional"                                                                             evaporation method                                               method by emulsion/                                                                        according to the                                                 evaporation  invention                                              ______________________________________                                        Distribution of                                                                           By suspension  By dissolution by                                  the peptide in             means of a third                                   the PLGA phase:            solvent (DMA)                                      Level of incor-                                                               poration of cal-                                                              citonin in the                                                                PLGA microspheres                                                             Example 3-1:                                                                              29.8%          79.4%                                              50/50 PLGA of                                                                 Mw (sic) = 50,000                                                             n = 0.5                                                                       Example 3-2:                                                                              39.7%          83.8%                                              50/50 PLGA of                                                                 Mw (sic) = 91,000                                                             n = 0.7                                                                       Example 3-3:                                                                              52.6%          92.4%                                              50/50 PLGA of                                                                 Mw (sic) = 115,000                                                            n= 0.8                                                                        "Burst" effect ob-                                                            served after the                                                              first day of release                                                          in vitro                                                                      Example 3-1:                                                                              12%            5%                                                 Example 3-2:                                                                              30%            3%                                                 Example 3-3:                                                                              11%            5%                                                 ______________________________________                                    

The viscosity of the polymer is represented by the letter n. It islinked to the molecular mass Mw (sic) by the Kuhn-Mark-Houwinkrelationship: n=K M^(-a) where K and a are constants for apolymer-solvent system at a fixed temperature.

EXAMPLE 4 Kinetics of Release--Principal Adjustable Parameters

1) Choice of the polymer

The polymer of composition 50/50 is more rapidly degraded than thecopolymers having other proportions of lactic acid and glycolic acid.(See reference: Miller R. A., Brady J. M. and Cutright D. E.--J. Biomed.Mater. res. (sic), 11, 711, 1977).

The intrinsic viscosity of the PLGA which is moreover mainly dependenton the size of the polymers is also involved in the kinetics of release.Thus, for calcitonin microspheres prepared with 50/50 PLGAs of differingviscosities., a release in vitro of calcitonin shows that the higher theintrinsic viscosity of the PLGA (the higher the molecular mass of PLGA)the slower is-the release of calcitonin into the external medium. Thus,for an incorporation of 50/50 PLGA of viscosity equal to 0.4 (MWmeasured by GPC: 36,000), 0.5 (50,000) or 0.7 (91,000), the percentageof calcitonin released in vitro after 14 days is 79.5, 75.5 or 43.2%respectively.

2) Size of the microspheres

Example 4a: Influence of the size of the microspheres on the release ofthe active ingredient

Calcitonin microspheres were prepared according to the followingprocedure.

Materials

Polymer phase:

    ______________________________________                                        Polymer phase:                                                                50/50 PLGA polymer n = 0.8                                                                           2.5 g                                                  solvent for the PLGA: CH.sub.2 Cl.sub.2                                                              20.0 g                                                 peptide: CTs (salmon calcitonin)                                                                     10.0 mg                                                level of charge of 0.4%/PLGA                                                  .sup.125 I-labelled peptide: .sup.125 I-CTs                                                          2.39 10.sup.6 cpm                                      ballast: lactose       5.0 mg                                                 level of charge of 0.2%/PLGA                                                  "third solvent": DMA   2.5 ml                                                 Aqueous phase:                                                                10 mM phosphate buffer, pH 8                                                                         75.0 ml                                                with 4% of gelatin (= emulsifier)                                             ______________________________________                                    

Method:

The peptide, the tracer and the ballast are solubilized in DMA.

They are then added to the polymer in solution in CH₂ Cl₂ in order toform a homogeneous phase, the polymer phase.

Emulsion: the aqueous phase is then superposed on the polymer phase andmixed with the polymer phase by means of a helix revolving at 1,500revolutions/min for one minute.

Evaporation: a stirring of 500 revolutions/min is maintained for ±12hours, at room temperature, with suction of air at the surface of theemulsified mixture.

The emulsion is then diluted 2-fold in water-and stirred for 1 hour, at37° C.

The microspheres are recovered in water on successive sieves ofprogressively smaller meshes, 200-100-50-20 microns.

They are then dried for 24 hours in an oven under vacuum, at roomtemperature.

They are then irradiated at 2.5 Mega Rad.

Results:

Size of the microspheres obtained:

    ______________________________________                                        Size (x)                                                                              25<x<50μ                                                                             50<x<100μ                                                                             100<x<200μ                                                                          x>200μ                               in microns                                                                    Percentage                                                                            7.5%      39.3%      46.2%    0.7%                                    by weight                                                                     of the mi-                                                                    crospheres                                                                    ______________________________________                                    

The level of incorporation of calcitonin into the microspheres: it ismeasured by means of the tracer (¹²⁵ I-CTs) introduced in thepreparation and by taking into account the yield by weight of themicrospheres relative to the weight of the polymer employed. A total of85.1% of CTs incorporated is found.

Release "in vitro" of calcitonin: 200 mg of microspheres are placed in10 ml of 10 mM phosphate buffer, pH 7 containing 0.5% of BSA at 37° C.in an oscillating bath.

    ______________________________________                                        Size of the                                                                   microspheres 25-50μ 50-100μ                                                                             100-200μ                                   ______________________________________                                        After:  1     day    11.2%   3.2%   2.8%                                              3     days   16.0%   4.5%   4.0%                                              4     days   16.5%   5.0%   4.7%                                              7     days   19.8%   5.5%   4.9%                                              10    days   25.2%   7.4%   6.4%                                              14    days   31.4%   7.7%   7.0%                                              25    days   73.0%   31.6%  31.0%                                             28    days   100.0%  40.2%  39.0%                                             35    days   --      64.5%  56.0%                                             40    days           89.0%  79.0%                                             42    days           100.0% 89.0%                                             45    days           --     100.0%                                    ______________________________________                                    

Example 4b: Parameters influencing the size of microspheres

Example 4b-I: rate of emulsion

50/50 PLGA: 2.5 g dissolved in CH₂ Cl₂ :20 g Salmon calcitonin: 5 mgdissolved in 2.5 ml of DMA and then added to the polymer phase.

Aqueous phase: 75 ml of 5% gelatin (weight/volume) in water.

The 2 phases are emulsified

either by stirring with a helix at 2,000 revolutions/minute for 1minute,

or by stirring with a helix at 500 revolutions/ minute for 1 minute.

Evaporation of the CH₂ Cl₂ and the passage of DMA in water are carriedout with continuous stirring of 500 revolutions/minute.

After several washes in water, a sample of microspheres is analyzed inthe "Fritsch particle sizer" (Analysette 22).

Results

    ______________________________________                                                         Emulsion at                                                                              Emulsion at                                       Percentage of the micro-                                                                       2,000 r/min                                                                              500 r/min                                         spheres (by weight)                                                                            Size of the                                                                              Size of the                                       having a size less                                                                             microspheres                                                                             microspheres                                      than             (μ)     (μ)                                            ______________________________________                                        10% <            16.4       78.9                                              30% <            35.2       164.0                                             50% <            52.3       212.0                                             70% <            73.0       271.0                                             90% <            126.0      351.0                                             ______________________________________                                    

Example 4b-II: The concentration of the emulsifier

50/50 PLGA: 2.5 g dissolved in CH₂ Cl₂ : 20 g Salmon calcitonin: 5 mgdissolved in 2.5 ml of DMA and then added to the polymer phase.

Aqueous phase:

either: 75 ml of 5% gelatin (weight/volume) in water,

or: 75 ml of 1% gelatin (weight/volume) in water.

The two phases are emulsified by stirring with a helix at 2,000revolutions/minute for 1 minute.

The evaporation of CH₂ Cl₂ and the passage of the DMA in water arecarried out with a stirring of 500 revolutions/minute with a helix.

After several washes in water, the microspheres are analyzed in the"Fritsch Particle Sizer" (Analysette 22).

Results

    ______________________________________                                                         Emulsifier:                                                                              Emulsifier:                                       Percentage of the micro-                                                                       5% gelatin 1% gelatin                                        spheres (by weight)                                                                            Size of the                                                                              Size of the                                       having a size lower                                                                            microspheres                                                                             microspheres                                      than             (μ)     (μ)                                            ______________________________________                                        10% <            16.4       37.4                                              30% <            35.2       73.6                                              50% <            52.3       104.0                                             70% <            73.0       140.0                                             90% <            126.0      215.0                                             ______________________________________                                    

3) Choice of the ballast

The presence or the concentration of ballast in microspheres as well asthe nature of the ballast-may influence the kinetics of release ofcalcitonin as shown by the following experiment:

Materials:

Polymer phase:

50/50 PLGA: 2.5 g dissolved in CH₂ Cl₂ : 20.0 g

salmon calcitonin: 10 mg dissolved in: DMA: 2.5 ml

ballast:

    ______________________________________                                        A            B          C                                                     ______________________________________                                        Without      Lactose 0.2%                                                                             Gelatin 0.2%                                          ______________________________________                                    

Aqueous phase:

10 mM phosphate buffer, pH 8 75 ml with 4% of gelatin (weight/volume)

Method:

The two phases are emulsified by stirring with a helix at 1,500revolutions/minute for one minute.

A stirring of 500 revolutions/minute is then maintained until completeevaporation of the CH₂ Cl₂ and passage of the DMA in the aqueous phase.

After several washes with water, the microspheres are recovered onsieves and dried under vacuum.

Results:

Release "in vitro" obtained in the same conditions as in point 2) ofExample 3.

    ______________________________________                                                 Cumulative percentage of CTs released                                         A       B                                                                     without (+0.2%    C                                                           ballast lactose)  (+0.2% gelatin)                                    ______________________________________                                        After:                                                                              1     day    2.0%    2.8     6                                                2     days   2.2%    4.0     8                                                7     days   3.4%    5.0     11                                               12    days   4.3%    7.4     15                                               20    days   16.0%   22.0    36                                               25    days   25.0%   31.0    46                                               30    days   32.0%   41.0    59                                               35    days   39.0%   56.0    83                                               37    days   44.0%   66.0    88                                               40    days   59.0%   79.0    100                                              42    days   69.0    100.0   --                                               48    days   100.0   --      --                                         ______________________________________                                    

4) Level of charge of peptide

Materials

Polymer phase:

50/50 PLGA: 0.5 g dissolved in: CH₂ Cl₂ : 10 g

DMA=3 ml in which is dissolved salmon calcitonin in the followingamount:

    ______________________________________                                        A                  B                                                          ______________________________________                                        10 mg              25 mg                                                      Level of charge of 2%                                                                            Level of charge of 5%                                      ______________________________________                                    

Ballast: without Aqueous phase:

10 mM phosphate buffer, pH 8 with 4% gelatin (weight/volume): 75 ml.

Method

Release "in vitro" obtained in the same conditions as in point 2) ofExample 3.

    ______________________________________                                                      Cumulative percentage of                                                      CTs released                                                    Level of charge A         B                                                   of salmon calcitonin                                                                          2%        5%                                                  ______________________________________                                        After:  1     day       3.0%    6.5%                                                  4     days      4.8%    13.0%                                                 6     days      5.7%    16.0%                                                 10    days      7.4%    18.5%                                                 15    days      8.6%    23.0%                                                 21    days      9.7%    27.0%                                         ______________________________________                                    

EXAMPLE 5 Reduction of the Residual Level of Toxic Solvent in theMicrospheres

Materials

50/50 PLGA: 1 g dissolved in 7 g of CH₂ Cl₂.

Salmon calcitonin: 20 mg dissolved in 2 ml of DMA and then added to thepolymer phase.

Aqueous phase:

a.*either: 100 ml of 5% gelatin (weight/volume) in 10 mM phosphatebuffer, pH 8 (sic).

b.*or: a mixture of 80 ml of 5% gelatin (weight/volume) in 10 mMphosphate buffer, pH 8 (sic)+20 ml of ethanol.

Method

The two phases are emulsified by means of a helix revolving at 1,500revolutions/minute, for 1 minute.

A stirring of 500 revolutions/minute for 12 hours at 37° C. with suctionof air enables the evaporation of CH₂ Cl₂ and the passage of DMA towardsthe aqueous phase.

The microspheres are recovered on successive sieves of 200 and 25microns.

They are then dried for 24 hours in an oven under vacuum at roomtemperature and then irradiated at 2.5M Rad.

Results

The levels of residual dichloromethane in the dry microspheres (fractionof 25 to 200 microns) are determined by aqueous phase chromatography.These levels are expressed as percentage of solvent relative to the dryweight of the microspheres.

    ______________________________________                                        Method of preparation                                                                          Residual level of CH.sub.2 Cl.sub.2                          ______________________________________                                        a.    without ethanol in the                                                                       1.36%                                                          aqueous phase                                                           b.    with 20% of ethanol in                                                                       0.14%                                                          the aqueous phase                                                       ______________________________________                                    

EXAMPLE 6 Adaptation of the Technique to Other Polypeptides

The application of the technique is conditioned by the solubility of thepolypeptides in the third solvent. This property is common to numerouspolypeptides, as has been shown for calcitonin.

Example:

LHRH ("Luteininizing Hormone Releasing Hormone") is soluble at not lessthan 50 mg/ml in DMA.

HGH ("Human Growth Hormone") is soluble at 2.6 mg/ml of DMA.

In order to increase the solubility of certain polypeptides which areless soluble in the "third solvent", it is possible to add an aqueoussolution of the peptide to the "third solvent" which is of coursemiscible with water.

Accordingly, it has been possible to incorporate the following peptidesin the PLGA microspheres by the technique according to the invention:calcitonin, hGh (sic), bGH (sic), ANF, somatostatin, LHRH, insulin,somatomedin C, ACTH, ADH, TPA as well as various cytokines:erythropoietin, GMCSF, GCSF, IL3, IL1, IL6, IL2, IL4, TNF, alphainterferon, beta interferon, beta TGF, PDGF and EGF.

The table below collates the levels of incorporation obtained for thevarious peptides using the "standard" procedure described belowaccording to the invention without individual optimization for eachpeptide. The values therefore represent a minimum level ofincorporation. The standard procedure used is as follows:

Materials

50/50 PLGA: 2.5 g dissolved in 20 g of CH₂ Cl₂. Third solvent: DMA: 2.5ml in which the active ingredient is dissolved.

Aqueous phase:

75 ml of 4% gelatin (weight/volume) in 10 mM phosphate buffer, pH 8.

Method

The polymer phase in which the active ingredient is dissolved by meansof the third solvent, is emulsified with the aqueous phase by a helixstirred at 1,500 revolutions/minute and this for one minute.

A stirring of 500 revolutions/minute is maintained at room temperaturewith suction of air in order to facilitate the evaporation of CH₂ Cl₂and the passage of the third solvent in the water.

The microspheres are then recovered on a sieve after having beenabundantly washed in water.

They are then dried at room temperature under vacuum.

    ______________________________________                                        Active ingredient                                                             incorporated in the                                                                            Percentage of the                                            microspheres     peptide incorporated                                         ______________________________________                                        hGH (sic) (human 77.0%                                                        growth hormone)                                                               Somatomedin C (IGF-1)                                                                          52.0%                                                        TPA (Tissue      96.0%                                                        Plasminogen Activator)                                                        Insulin          98.0%                                                        IL4              77.5%                                                        IL6              43.8%                                                        Erythropoietin   40.0%                                                        TNF              66.4%                                                        Salmon calcitonin                                                                              92.4%                                                        Human calcitonin 85.0%                                                        IL2              78.5%                                                        GRH              84.0%                                                        LHRH             75.0%                                                        GMCSF            59.0%                                                        PTH (1-34)       54.0%                                                        Somatostatin     82.0%                                                        ANP (or ANF)     72.0%                                                        Alpha interferon 77.0%                                                        ______________________________________                                    

We claim:
 1. A process for preparing microparticles of the microspheretype of a water-soluble polypeptide or a pharmaceutically acceptablesalt thereof and a biocompatible and biodegradable polymer, comprisingthe steps of:a) dissolving a biocompatible and biodegradable polymer ina first volatile organic solvent immiscible with water, b) separatelydissolving water-soluble polypeptide in a second solvent which isnonvolatile, miscible with said first solvent, a solvent for saidpolymer, and miscible with water, c) mixing the solution of saidpolypeptide and the solution of said polymer to produce an organic phasecontaining said polymer and said polypeptide, d) emulsifying saidorganic phase in an immiscible dispersant aqueous phase containing anemulsifying agent, e) removing the two solvents from the microspheresbeing formed, with stirring, the first solvent being removed byevaporation, the second solvent as well as part of the first solventwhich is miscible therewith being removed by passage towards the aqueousphase by a mechanism of phase separation, and f) after removal of thesolvents, recovering the microspheres formed, optionally after washingin water and sieving.
 2. Process according to claim 1, wherein saidaqueous dispersant phase comprises a third solvent which is misciblewith water and miscible with said first solvant.
 3. Process according toclaim 1, wherein said first solvant is dichloromethane and said secondsolvent is dimethylacetamide.
 4. Process according to claim 2, whereinsaid third solvent is ethanol.
 5. Process according to claim 1, whereinsaid polymer is a lactic acid and glycolic acid copolymer.
 6. Processaccording to claim 1, wherein said polymer is a lactic acid and glycolicacid copolymer which comprises a proportion between the lactic acid andglycolic acid units of 40:60 to 75:25 respectively.
 7. Process accordingto claim 1, wherein said concentration of emulsifying agent in theaqueous phase is between 0.1 and 10% (weight/volume).
 8. Processaccording to claim 1, wherein the second nonvolatile solvent is selectedfrom the group consisting of dimethylacetamide, tetrahydrofuran,dioxane, dimethyl sulfoxide and dimethylformamide.
 9. The process ofclaim 1, wherein in at least one of steps (b) or (c), a ballast is addedwhich is dissolved or suspended in the organic polymer phase resultingfrom step (c).